Limited-proteolysis by serine and cysteine proteases plays a central regulatory role in many physiological and pathophysiological processes including coagulation, fibrinolysis, complement fixation, apoptosis, angiogenesis, tumor cell attachment and growth. Serpins (Serine Protease Inhibitors) are the principle protease inhibitors in human plasma (serpins make up 10% of plasma proteins on a molar basis), and have been shown to regulate these physiological processes. Well known examples include antithrombin which regulates the blood coagulation cascade; C1-inhibitor which controls complement activation; plasminogen activator inhibitors (PAI-1 and α2-antiplasmin) which regulate fibrinolysis; and alpha1-antitrypsin, also called alpha1 proteinase inhibitor, which modulates connective tissue remodeling (see Gils & Declerck, 1998 and Whisstock et al. 1998 for reviews on these and other serpins).
Serpins regulate proteases through a suicide-substrate inhibition mechanism forming a covalent complex between the protease active site serine and the bait P1 amino acid (Huntington et al., 2000). This results in a 1:1, SDS-resistant complex. In addition to the inhibitory serpins, there are also serpin family members that function as hormone transport or growth factor proteins (examples include the thyroxin and corticosteroid binding globulins and the vasopressor peptide source angiotensinogen). In addition, serpins have demonstrated involvement in angiogenesis and tumor growth (see for example O'Reilly et al., 1999).
As a class, serpins contain conserved residues located internal to the protein or on surface niches, therefore the serpin family has remarkably-conserved 3-dimentional structure despite less dramatic overall homology elsewhere (see Huber & Carrell,1989 and Whisstock et al., 1998 for reviews). Protease inhibitory serpins (of which the present invention relates) contain a mobile reactive site loop (P16-P10′) that is subject to proteolytic cleavage. This loop cleavage site (sometimes referred to as the “bait region”) is cleaved by a cognate protease. The loop region is flexible and has the ability to profoundly change its conformation (alanine-rich regions in the loop P13-P9 allow increased flexibility (Gils & Declerck, 1998)). Once cleaved, the P14-P1 portion of the loop has high affinity for insertion into a β-sheet domain (Whisstock et al., 1998; Huntington et al., 2000). In general, peptides corresponding to the P14-P1 or P14-P7 or P14-P10 loop regions and small molecule mimics of the loop region prevent protease inhibitory activity.
The serpin proteolytic cleavage site is designated P1P1′. It is generally accepted that the P1 residue is the major determinant of the protease specificity of the serpin (Gills & Declerck, 1998). Typically the bait region (especially the P1 site) mimics the natural substrate of the cognate protease. While each inhibitory serpin shows some preference for selected proteases, most serpins recognize several proteases with varying degrees of specificity (for example antithrombin will inhibit Factors IXa, Xa, XIa, as well as thrombin; α1-antitrypsin will inhibit trypsin and neutrophil elastase; PAI-1 inhibits tPA, uPA and trypsin etc.). Furthermore, relatively minor amino acid changes in the bait region, especially at the P1 position, can dramatically change the specificity of the serpin (Gils and Declerck, 1998).
In addition to the bait region, domains away from the cleavage site also confer selectivity. For some serpins, non-enzymatic cofactors activate the serpin and modulate the rate of protease inactivation. For example antithrombin, PAI-1, protease nexin 1, protein C inhibitor and heparin cofactor II are stimulated by heparin. Furthermore, the specificity of a serpin may depend on the cofactor composition and concentration. For example the specificity of antithrombin depends on the size of the heparin molecule. Large molecular weight heparins stimulate inhibition of thrombin where as low molecular heparin has more FXa selectivity. Another example is vitronectin, which binds to PAI-1 and increases its affinity for thrombin. Studies (Shirk, et al, 1994, Lane et al, 1994, Pratt et al 1991, Blinder, et al, 1989, Blinder et al 1991, Whinna et al, 1991, Ragg et al, 1991) have implicated one or more of the A, D and H helices of serpins (for example, the helices in antithrombin, protein C inhibitor and heparin cofactor II) which are rich in basic residues and bind heparin, and, 3-dimensional structures and models have been useful in the design of low molecular weight molecules which modulate serpin reactivity.
Recently, the crystal structure of a serpin-protease complex was reported (Huntington, et al, 2000) in which the reactive loop has been cleaved at the bait position. The tethered protease moves 70 angstroms, with the tether sequence (P10-P1) inserted in the “A” beta sheet of alpha anti-trypsin. This shallow groove, which we call the cleaved reactive loop-binding region, represents a potential target for small molecule inhibition.
Serpins in Disease and Therapy
Members of the serpin family play a variety of roles in physiology, disease and in therapy. Deficiencies in some serpins can cause well-characterized diseases. Alpha-1-antitrypsin deficiency, which is a common autosomal recessive disorder, is associated with development of emphysema, liver cirrhosis and hepatocellular carcinoma. Patients deficient in antithrombin are prone to sever thrombotic consequences.
Serpins are useful as therapeutics. Antithrombin is a potent inhibitor of thrombin-mediated vascular injury in the microcirculation in severe sepsis. This endogenous anticoagulant is rapidly depleted in the early phases of sepsis as a result of decreased synthesis, increased destruction, and enhanced clearance by thrombin-antithrombin complex formation. The therapeutic efficacy of antithrombin in experimental sepsis is readily demonstrable in numerous animal systems (Opal 2000).
In addition to the therapeutic activity of the serpins, modulators of serpin activity have well documented utility in antithrombotic therapy. The anticoagulant efficacy of heparin and low molecular weight heparin is mediated by antithrombin inhibition of proteases (i.e. thrombin and FXa). Indeed antithrombin is required for the anti-coagulant efficacy of heparin and low molecular weight heparins which are useful in the treatment of arterial thrombosis (for example myocardial infarction, unstable angina & stroke) and venous thrombosis (for example deep vein thrombosis and pulmonary embolism).
Inhibitors of serpins have also shown utility. Antibodies or small molecule inhibitors can inhibit serpin activity and prolong cognate protease function. Examples of serpin inhibition with therapeutic value that preserve fibrinolytic function include antibodies to alpha 2-antiplasmin (Reed et al., 1990) and plasminogen activator inhibitor-1 (Biemond et al., 1995). Both have been shown to improve thrombolysis under thrombotic conditions in animal models of disease.
Serpins are also useful in measurement of cognate protease activation and are useful in diagnosis of disease states. In general, proteolytic enzymes form stable complexes with serpins that can be followed by measuring the cognate protease-serpin complex using ELISA or other techniques. As such, measurement of the serpin-protease complex is useful as a reporter of cognate protease activation. An example is thrombin-antithrombin complex (TAT) which is a reporter of thrombin activation in vivo. TAT levels are elevated in thrombotic disease states and measurement of thrombin-antithrombin complexes is useful in the diagnosis of arterial and venous thrombotic diseases as well as a biomarker of disease and therapy. In general, serpin inhibition of proteolytic activities are measured by following the inhibition of cognate protease in vitro or in vivo.
Using the above examples, it is clear the availability of a novel cloned serpin provides opportunity for adjunct or replacement therapy, and are useful for the identification of serpin agonists, or stimulators (which might stimulate and/or bias serpin action), as well as, in the identification of serpin inhibitors. All of which might be therapeutically useful under different circumstances. The serpin of the present invention can also be used as a scaffold to tailor-make specific protease inhibitors and prevent angiogenesis. In addition detection of the serpin-cognate protease complex can be a useful diagnostic tool.
Polynucleotides and polypeptides corresponding to a portion of the full-length LSI-01 polypeptide of the present invention, in addition to its encoding polynucleotides, have been described by Baker et. al., International Publication Number WO 00/12708, gene UNQ692. Baker et al. did not appreciate the fact that their UNQ692 gene was not representative of the entire coding region, nor the fact that the UNQ692 translation product was deficient in the N-terminal domain. Baker et al., have termed their peptide fragment, encoded by their UNQ692 polynucleotide fragment, as PRO1337. Baker et al. teach the PRO1337 peptide as representing a homologue of a human thyroxin-binding globulin (TBG; Genbank Accession No. gi|4507377), and did not appreciate the fact that the protein shares higher identity to Arg/lys protease inhibitor serpins in domains essential for serpin activity and specificity, as opposed to the hormone-binding serpins.
The inventors of the present invention describe herein, the polynucleotides corresponding to the full-length LSI-01 gene and its encoded polypeptide. Also provided are polypeptide alignments illustrating the strong conservation of the LSI-01 polypeptide to known protease inhibitor serpins, and the dissimilarity of the protein in key amino acid domains to TBG. As inferred above, the dissimilarity is particularly evident in the ‘bait’ region, and the flexible loop region, which are essential for determining serpin function and specificity, as shown in FIG. 2. Based on this strong conservation, the inventors have ascribed the LSI-01 polypeptide as having at least some serpin protease inhibitory activities, particularly inhibition of proteases exhibiting Arg/lys specificity. Data is also provided illustrating the unique tissue expression profile of the LSI-01 polypeptide in lymphoid tissues, which has not been appreciated heretofore.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of LSI-01 polypeptides or peptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the LSI-01 polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.